PROJECT SUMMARY Cow?s milk allergy is among the most common pediatric food allergies, affecting about 2% of young children in the United States 1, 2, and its prevalence continues to increase 3. Although milk allergy is commonly outgrown later in life, it still poses a great health risk as avoidance of all dairy-containing products is difficult and accidental ingestion is common. Cow?s milk allergic reactions can be very severe, and are estimated to be responsible for up to 13% of fatal food-induced anaphylaxis 4. Diagnosis of milk allergy is challenging; tests relying on detection of milk allergen-specific IgE antibodies have a high rate of false positives, and a non- negligible rate of false negatives. Food challenges provide the most accurate diagnostic tool in the clinic, but require significant resources and highly trained staff to be carried out with acceptable risk, which is not always available outside of well-controlled research settings. To improve diagnostics of milk and other food allergies, the underlying disease mechanisms need to be better understood. In addition, the rate at which milk allergy is outgrown is patient dependent and in some cases, this allergy is still present in adulthood. Aside from milk- specific IgE titers, which are not reliably accurate in predicting food challenge outcomes, there are no available biomarkers to assess if a patient will outgrow their allergy in coming months and when they can begin to safely ingest dairy or raw milk. As CD4 T cells are known to play a key role in mediating food allergy 5, our central hypothesis is that studying the frequency and phenotype of milk allergen-specific T cells in cohorts with different disease manifestations will define molecular markers of disease status and progression to tolerance, and this information could be utilized for developing improved and new diagnostic tests. Accordingly, we propose to comprehensively define T cell epitopes from milk allergens (Aim 1) and use these as reagents to determine the phenotype of antigen-specific T cells from donors with different clinical manifestations (Aim 2), as well as track the longitudinal development of antigen-specific T cells during the disease course (Aim 3). By comparing and contrasting phenotypic markers of milk allergen-specific T cells identified in this project with fungal, cockroach and mouse allergen-specific T cells defined in Projects 1 and 3, we will determine pathogenic features of allergen-specific T cells that are either specific to milk (or more broadly food) allergies or shared across other allergens.